The present invention relates to a method and system for characterizing lipoproteins in a sample, and more particularly, for characterizing lipoproteins in blood using light scattering and absorption properties of lipoproteins.
Absorption spectroscopy and light scattering methods are typically used to determine the composition and/or molecular weight of macromolecules in solution. Until recently absorption and light scattering have been considered to be separate and independent measurements even though the same physical phenomena often occur in practice for both techniques.
Absorption and light scattering measurements are usually conducted using electromagnetic energy having wavelengths in the ultraviolet to visible portion of the electromagnetic spectrum. The absorption and scattering data are typically collected by taking electromagnetic energy measurements from multiple observation angles. The theoretical background associated with absorption spectroscopy and light scattering methods is described in U.S. Pat. Nos. 6,330,058, 5,808,738 and 5,589,932, which are incorporated herein by reference. See also Alupoaei CE, et al., Biosens Bioelectron. 19, 893-903 (2004) and Alupoaei CE, et al., Biotechnol Bioeng. 86, 163-7 (2004).
Lipoproteins in blood are a heterogeneous group varying in size, composition and density. A typical lipoprotein includes a hydrophobic lipid core and a hydrophilic shell to which small proteins and other molecules are attached (usually via non-covalent interactions). The hydrophobic lipid core consists primarily of cholesterol ester and triglyceride while the hydrophilic shell consists primarily of free cholesterol and phospholipids.
Lipoproteins perform the important physiological function of delivering cholesterol and triglycerides through the endothelial lining of blood vessels to the sub-endothelial space such that the lipid components of lipoproteins form an important structural component within blood vessels. The lipoproteins circulate as small (e.g., 5 to 100 nm) insoluble particles of various densities. The densities of the lipoproteins are typically classified as very low density (VLDL), low density (LDL) and high density (HDL), although other densities are known and have been classified.
Recent medical research has shown a strong correlation between heart disease and the abundance of small LDL particles within blood. The size as well as the density of the particles has been shown to be critically important because when localized in sub-endothelial space small LDL particles can activate a strong macrophage response that leads to the formation of a therosclerotic plaque. These small LDL particles, not simply the cholesterol and triglycerides themselves, are one primary cause of coronary vascular disease.
The analysis of high and low density lipoproteins (HDL and LDL), cholesterol and triglycerides in human blood has become an important diagnostic parameter in determining the risk of cardiovascular disease. Therefore, the clinical determination of HDL, LDL, total cholesterol and triglycerides in blood has received considerable attention due to the association with human health.
Some known methods need to separate lipoprotein particles in order to determine the concentrations of HDL, LDL, total cholesterol and triglycerides in blood. Example separation techniques include precipitation and/or centrifugation in combination with enzymes conjugated to an antibody. In some forms, precipitation reagents such as polyanion-divalent cations are used to separate the VLDL and LDL from the HDL. The HDL is then analyzed via enzymatic methods and separate measurements are taken for total cholesterol and total triglyceride in order to determine LDL by an indirect approach.
There are some recent methods that have been developed using antibodies to directly determine LDL. However, these methods still require particle separation.
There are methods that do not require particle separation. As an example, a diagnostic procedure for determining a lipoprotein size and density profile using nuclear magnetic resonance is available from LipoScience, Inc., Raleigh, N.C. (http://www.liposcience.com/).
A comprehensive description of cholesterol testing can be found in the NIH publication 95-3044: “National Cholesterol Education Program, Recommendations on Lipoprotein Measurement: September 1995, with cited references inclusive as background information.